Project Summary Despite advances in our understanding of the mechanisms mediating platelet thrombus formation, current antiplatelet drugs increase the risk of major bleeding. In an effort to identify novel therapeutic targets, we and others showed that intravascular protein disulfide isomerase (PDI) is crucial for full activation of ?IIb?3 integrin and platelet accumulation in arterial thrombosis, providing insights into a new antithrombotic agent. However, blocking the oxidoreductase activity of intravascular PDI prolongs tail bleeding times in mice. Our preliminary data have demonstrated that endoplasmic reticulum (ER) oxidoreductin 1? (Ero1?), a key oxidase of PDI in the ER, is released from activated platelets and that inhibition or deletion of platelet Ero1? alters the activity of platelet surface-bound PDI and impairs platelet aggregatory function. Furthermore, we have found that global deletion of Ero1? reduces the size of platelet thrombus formation without affecting initial platelet adhesion and tail bleeding times following vascular injury. Using biochemical, cellular and in vivo animal studies with novel pharmacological inhibitors and Ero1? conditional knockout and global knockout mice developed by our lab, we will test the hypothesis that platelet-released Ero1? regulates the function of PDI and ?IIb?3 integrin on the cell surface and contributes to the propagation step of platelet thrombus formation without affecting hemostasis following vascular injury. In Aim 1, we will identify the molecular mechanism by which extracellular Ero1? promotes platelet aggregation. In Aim 2, we will test whether arterial thrombotic conditions alter the function of extracellular Ero1?. In Aim 3, we will determine the pathophysiological role of intravascular and platelet-derived Ero1? in platelet adhesion and accumulation and vessel occlusion under thrombotic conditions. The proposed studies will identify a central regulatory mechanism of platelet thrombus formation and lead to the discovery of novel therapeutic strategies for the safe and effective treatment of thrombotic disease.